Oligonucleotide serial synthesis apparatus and method for synthesizing oligonucleotides using the same

Abstract

An oligonucleotide continuous synthesis device according to the present invention comprises: a starting material container for storing a blocked protected nucleoside that is a starting material; a first reagent container for storing a reagent used for chain extension of the blocked protected nucleoside; a second reagent container for storing a reagent for a reaction for oxidizing or sulfurizing the phosphate linkage of the extended nucleotide obtained by the chain extension; and a third reagent container for storing a reagent for a reaction for deprotecting the oxidized or sulfurized extended nucleotide. In this device, the starting material, the first reagent, and the second reagent are supplied continuously, thereby performing oligonucleotide synthesis in a flow system.

Description

Technical Field

[0001] The present invention relates to an apparatus for continuously synthesizing oligonucleotides in a liquid-phase flow system and a method for synthesizing oligonucleotides using the same.

Background Art

[0002] [[ID=第十二]]In recent years, the attention to nucleic acid drugs based on natural or modified oligonucleotides as a basic skeleton has been increasing. In order to obtain a nucleic acid drug designed to achieve a desired action, a chemical synthesis method is widely used in the synthesis of oligonucleotides.

[0003] For the chemical synthesis of oligonucleotides having a length of around 20 to 50 mers, there are mainly a solid-phase synthesis method and a liquid-phase synthesis method. As a synthesis apparatus used in the solid-phase synthesis method, a solid-phase synthesis apparatus exemplified in Patent Document 1, in which one end is fixed to a support carrier and the chain elongation of oligonucleotides is advanced step by step by repeatedly performing treatments with raw materials and reactants, is widely used. The phosphoramidite method is mainly used for the synthesis in the solid-phase synthesis apparatus, and as the support carrier, controlled pore glass (CPG) and porous polystyrene are known.

[0004] As an example of the liquid-phase synthesis method of oligonucleotides, Patent Document 2 proposes a method of using a nucleoside protected body in which polyethylene glycol (PEG), a hydrophilic polymer, is introduced into the base portion of a nucleoside as a starting material or a building block to facilitate the separation and purification of intermediate products and synthesize oligonucleotides.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] Solid-phase synthesis using a solid-phase synthesizer can easily achieve chain elongation of nucleic acid oligomers, but because the reaction takes place on a support within the column that serves as the reaction vessel, scaling up is difficult. In addition, solid-phase synthesis requires the use of excess reagents at each stage, making it costly when large quantities of oligonucleotides are needed. Specifically, performing solid-phase synthesis on a scale exceeding 1 mmol using a CPG support requires considerable expense.

[0007] From the above perspective, liquid-phase synthesis methods that can use commonly available reactors are advantageous for synthesizing nucleic acid oligomers on a large scale. Even with liquid-phase synthesis methods using such reactors, many steps are required to obtain the target product when synthesizing oligonucleotides in quantities exceeding 100 g per batch, for example.

[0008] Specifically, liquid-phase batch synthesis methods include chain extension, deprotection, removal of excess reagents and by-products, purification, and desalting. For large-scale synthesis, each step requires a corresponding reactor and purification apparatus. Since multi-step synthesis generates impurities such as unreacted reagents and reaction by-products, and purification by methods such as column chromatography is necessary to remove them, process improvements and cost reductions are required for the production of large quantities of oligonucleotides.

[0009] Furthermore, in order to manufacture nucleic acid active pharmaceutical ingredients (APIs) to GMP (Good Manufacturing Practice) standards, each of the above processes must be installed in a separate cleanroom. This requires not only enormous management and maintenance costs for each piece of equipment, but also the construction costs of large facilities, as well as a vast and clean space. [Means for solving the problem]

[0010] This invention has been made in view of these circumstances, and aims to provide a liquid-phase flow type continuous oligonucleotide synthesis apparatus and a method for synthesizing oligonucleotides using the same, which enable large-scale synthesis of oligonucleotides with a simpler apparatus and fewer steps.

[0011] To solve the above problems, the liquid-phase flow type oligonucleotide continuous synthesis apparatus and the oligonucleotide synthesis method using the same of the present invention employ the following means.

[0012] A first aspect of the present invention is a raw material container containing a mixture of at least two types of protective nucleoside block bodies, which are raw materials, A first reagent container containing reagents for a reaction to extend a nucleotide chain by reacting at least two of the aforementioned protected nucleoside block bodies with each other, A second reagent container for containing reagents for the reaction of oxidizing or sulfidizing the phosphate bond of the nucleotide elongated product obtained by chain elongation, A third reagent container for containing reagents for a reaction to deprotect nucleotide elongators that have been oxidized or sulfurized, A first joint connects the piping extending from the raw material container to the piping extending from the first reagent container, A first reaction tube connecting the first fitting and the second fitting connected to the piping extending from the second reagent container, A second reaction tube connecting the second fitting and the third fitting connected to the piping extending from the third reagent container, Equipped with, The raw materials, the first reagent, the second reagent, and the third reagent are each in solution. Each of the piping lines from the raw material container, the first reagent container, the second reagent container, and the third reagent container is connected to a liquid delivery device that delivers the solution from each container to the reaction tube connected via the piping. At the first joint, the raw material solution and the first reagent solution are combined to form a raw material mixture, which is then led into the first reaction tube. In the first reaction tube, a reaction for nucleotide chain elongation takes place using a mixture of starting materials, and the solution containing the chain elongation product is then introduced into the second reaction tube. In the second reaction tube, a reaction for oxidation or sulfidation of the chain extension product takes place. This invention provides a continuous oligonucleotide synthesis apparatus that enables the continuous synthesis of oligonucleotides by continuously supplying raw materials, a first reagent, and a second reagent.

[0013] In the first embodiment, a first waste liquid container for containing waste liquid containing waste reagents generated by the chain extension reaction, the oxidation or sulfidation reaction, and the deprotection reaction, The third joint and the separator connected to the piping extending from the first waste liquid container are connected to the third reaction tube. The deprotection reaction is carried out in the third reaction tube and led to the separator, where the crude oligonucleotide product obtained by the chain extension reaction and the oxidation or sulfidation reaction is separated from the waste reagents generated in each reaction, and the waste liquid containing the waste reagents is stored in the first waste liquid container.

[0014] In the above embodiment, a purification reagent container for containing a purification reagent for purifying the crude oligonucleotide product, A second waste liquid container for containing waste liquid containing waste reagents generated in the aforementioned purification process, Furthermore, A liquid delivery device is connected to the piping from the aforementioned purified reagent container, which delivers the solution from the purified reagent container through the piping. The separator, the piping extending from the purification reagent container, and the piping extending from the second waste liquid container are connected to a purification tank, the crude oligonucleotide product is purified in the purification tank, and the final oligonucleotide product obtained from the downstream side of the purification tank is contained in the product container, thereby carrying out the reaction for chain extension and the purification of the crude oligonucleotide product in a continuous liquid-phase flow format, and the final oligonucleotide product is synthesized continuously from the raw materials.

[0015] In the above aspect, the purification tank and the pipe extending from the product liquid container may be connected to a concentration and drying tank, and in the concentration and drying tank, the oligonucleotide product obtained by purification may be concentrated and dried, and the finally concentrated and dried oligonucleotide product may be stored in the product container.

[0016] In the above aspect, a monitoring device may be arranged at least at one location between the downstream side of the first reaction tube and the second joint and between the downstream side of the second reaction tube and the third joint.

[0017] In the above aspect, a monitoring device may be arranged between the downstream side of the separator and the purification tank.

[0018] In the above aspect, a monitoring device is arranged between the purification tank and the concentration and drying tank, and a joint for connecting to a deviation recovery device for recovering a deviation having a different length from the target oligonucleotide product is arranged between the monitoring device and the concentration and drying tank.

[0019] In the above aspect, the monitoring device may be selected from a mass spectrometer, NMR, UV-visible spectrometer, near-infrared spectrometer, Raman spectrometer, conductivity measuring device, and pH meter.

[0020] In the above aspect, it may be connected in a dividable manner on the downstream side of the monitoring device.

[0021] In the above aspect, two types of the protected nucleoside block bodies may be mixed and stored in the raw material container.

[0022] In the above aspect, three types of the protected nucleoside block bodies may be mixed and stored in the raw material container.

[0023] In the above embodiment, the chain extension step, oxidation or sulfurization step, deprotection step, and purification step may be carried out continuously in the liquid phase.

[0024] In the above embodiment, at least one of the first reaction tube and the third reaction tube may be a tube in which the catalyst used in the reaction is fixed inside.

[0025] A second aspect of the present invention is a method for the continuous synthesis of oligonucleotides using the oligonucleotide continuous synthesis apparatus according to the first aspect, comprising a chain elongation step of reacting at least two types of protected nucleoside block bodies with each other to elongate the nucleotide chain, An oxidation or sulfurization step in which the phosphate bond of the nucleotide elongation product obtained in the chain elongation step is oxidized or sulfurized, A deprotection step is performed on the nucleotide elongated product obtained in the oxidation or sulfidation step, A purification step to purify the crude oligonucleotide product obtained in the deprotection step, Equipped with, The present invention provides a method for synthesizing oligonucleotides, comprising a chain extension step, an oxidation or sulfurization step, a deprotection step, and a purification step, all carried out sequentially in a liquid phase.

[0026] In the second embodiment described above, the number of nucleosides contained in one type of protective nucleoside block used in the chain extension step may be in the range of 1 to 48. [Effects of the Invention]

[0027] The oligonucleotide continuous synthesis apparatus of the present invention enables large-scale oligonucleotide synthesis using a simpler apparatus and fewer steps. [Brief explanation of the drawing]

[0028] [Figure 1] This figure shows an overview of the configuration of the oligonucleotide continuous synthesis apparatus according to the first embodiment. [Figure 2] This figure shows an overview of the configuration of the oligonucleotide continuous synthesis apparatus according to the second embodiment. [Figure 3A] This figure shows an overview of the configuration of an oligonucleotide continuous synthesis apparatus according to a modified example 1 of the second embodiment. [Figure 3B] This diagram shows an overview of the configuration of Example 1 of the modularization of an oligonucleotide continuous synthesis apparatus. [Figure 3C] This diagram shows an overview of the configuration of Example 2 of the modularization of an oligonucleotide continuous synthesis apparatus. [Figure 4] This figure shows an overview of the configuration of an oligonucleotide continuous synthesis apparatus according to a modified example 2 of the second embodiment. [Figure 5] This figure shows one specific example of the configuration of an oligonucleotide continuous synthesis apparatus according to the second embodiment. [Figure 6] This figure shows one specific example of the configuration related to liquid delivery from the raw material container in the oligonucleotide continuous synthesis apparatus according to the second embodiment. [Figure 7] This diagram shows an overview of the connection section in a configuration of a continuous oligonucleotide synthesis apparatus according to the first or second embodiment, in which multiple raw material containers are arranged and pumped. [Figure 8] This diagram shows an overview of the connection section in a configuration of a continuous oligonucleotide synthesis apparatus according to the first or second embodiment, in which multiple raw material containers are arranged and pumped by gas. [Figure 9] This figure shows the HPLC chart of the crude 24-mer product obtained by carrying out each reaction in a reaction tube used in a continuous oligonucleotide synthesis apparatus. [Figure 10] This figure shows the HPLC chart of the fraction containing the target product, obtained by ultrafiltration membrane separation of the 24-mer obtained in Example 4. The upper panel shows the HPLC chart after purification, and the lower panel shows the HPLC chart of the filtrate. [Figure 11] This figure shows the HPLC chart of the fraction containing the target product, obtained by gel filtration purification of the 24-mer obtained in Example 4. [Figure 12] This figure shows the HPLC chart of the fraction containing the target product, obtained by ODS filtration of the 24-mer obtained in Example 4. [Figure 13] The upper panel shows the HPLC chart of the product obtained by ethanol precipitation in a deprotection solution containing ammonia, and the lower panel shows the HPLC chart of the product obtained by isopropyl alcohol (IPA) precipitation. [Modes for carrying out the invention]

[0029] An embodiment of the oligonucleotide continuous synthesis apparatus according to the present invention will be described below with reference to the drawings.

[0030] [First Embodiment] Hereinafter, a first embodiment of the present invention will be described with reference to Figure 1. Figure 1 shows an example of the configuration of the oligonucleotide continuous synthesis apparatus 100 according to this embodiment. The oligonucleotide continuous synthesis apparatus 100 is an apparatus that starts with raw materials and carries out various reactions in a liquid-phase flow format to continuously produce the target oligonucleotide final product.

[0031] The oligonucleotide continuous synthesis apparatus 100 is equipped with a raw material container 1, a first reagent container 2, a second reagent container 3, a third reagent container 4, a purified reagent container 5, and a target product container 6 as containers for various reagents. In addition, it is equipped with a first waste liquid container 7-1, a second waste liquid container 7-2, and a third waste liquid container 7-3 for storing waste reagents, by-products, and waste liquids generated in each reaction described later. In the example shown in Figure 1, the third reagent container 4 is configured to include third reagent containers 4-1 and 4-2, each containing a different type of reagent.

[0032] The oligonucleotide continuous synthesis apparatus 100 according to this embodiment further includes reaction tubes 21, 22, 23, and 24 that carry out different reactions inside, a separator 8 for separating waste reagents and by-products from the target product, a purification tank 9 for purifying the separated target product, and a freeze-drying tank 10 for freeze-drying the purified target product into powder.

[0033] The oligonucleotide continuous synthesis apparatus 100 according to this embodiment employs a liquid-phase flow system in which a solution containing reagents and reaction products is delivered in one direction from the raw material container 1 to the target product container 6, and the reaction proceeds to the next stage while the reaction takes place in the liquid phase.

[0034] The piping extending from the raw material container 1 and the piping extending from the first reagent container 2 are connected by the first joint 11-1.

[0035] The first joint 11-1 is connected to the second joint 11-2, which is connected to the piping extending from the second reagent container 3, via the first reaction tube 21, on the downstream side in the flow direction.

[0036] The second joint 11-2 is connected to the third joint 11-3, which is connected to the piping extending from the third reagent container 4-1, via the second reaction tube 22, on the downstream side in the flow direction.

[0037] The third joint 11-3 is connected to the third joint 11-4, which is connected to the piping extending from the third reagent container 4-2, via the third reaction tube 23, on the downstream side in the flow direction.

[0038] The third joint 11-4 is connected to a separator 8, which is connected to a pipe extending toward the first waste liquid container 7-1, on the downstream side in the flow direction, via the fourth reaction tube 24.

[0039] Separator 8 is connected to the purification tank 9 on the downstream side in the flow direction. The purification tank 9 is connected to piping extending from the purification reagent container 5, which contains the purification reagents supplied to the purification tank 9, and to piping extending from the second waste liquid container 7-2, which contains the waste reagents and by-products separated in the purification tank 9.

[0040] The purification tank 9 is connected to the freeze-drying tank 10 on the downstream side in the flow direction. The freeze-drying tank 10 is connected to a piping extending from a third waste liquid container 7-3, which contains the liquid produced by freeze-drying, and to a target material container 6, which contains the target material obtained by freeze-drying.

[0041] A method for synthesizing oligonucleotides using the oligonucleotide continuous synthesis apparatus 100 according to this embodiment will be described. The raw material container 1 contains two or three types of protected nucleoside block bodies, which are the raw materials. The protected nucleoside block bodies used in this embodiment will be described later.

[0042] The first reagent container 2 contains activators for reacting two or three types of protected nucleoside block bodies with each other to elongate nucleotide chains. Examples of activators in the first reagent container 2 include activators that activate the amidite moiety in the phosphoramidite method. Typical activators include, but are not limited to, 1H-tetrazole derivatives such as 1H-tetrazole and S-ethylthio-1H-tetrazole, imidazole derivatives such as dicyanoimidazole and dichloroimidazole, and salts of sulfonic acid with azole or tertiary amines.

[0043] The activator described above is dissolved in a solvent and stored as a solution in the first reagent container 2. As the solvent, acetonitrile, dichloromethane, THF, etc., which are commonly used in the phosphoramidite method, can be used, and a mixture of multiple types of solvents may also be used.

[0044] The second reagent container 3 contains reagents for the reaction to oxidize or sulfurize the phosphate bond of the nucleotide elongated product obtained by chain elongation. Examples of oxidizing agents include, but are not limited to, commonly used iodine pyridine aqueous solution, TBHP toluene solution, TMSOOTMS / TMSOTf dichloromethane solution, 2-butanone peroxide dichloromethane solution, and (R)-(10-camphorsulfonyl)oxaziridine acetonitrile solution. Examples of sulfiding agents include, but are not limited to, commonly used Beaucage reagent, bis(phenylacetyl) disulfide (PADS), xanthan hydride, [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazolin-3-thione (DDTT), 5-phenyl1,2-dithiazole-3-one, bis(3-(triethoxysilyl)propyl))tetrasulfide) / N-methylimidazole, propylene sulfide, etc.

[0045] Third reagent container 4 contains reagents for the reaction to deprotect nucleotide elongators that have been oxidized or sulfurized. In the example in Figure 1, since two types of deprotection reagents are used, third reagent containers 4-1 and 4-2 are provided.

[0046] Third reagent containers 4-1 and 4-2 contain reagents for removing protecting groups from the phosphate or nucleic acid base moieties. For example, one container can contain a reagent for deprotecting the phosphate moiety, while the other container can contain a reagent for deprotecting the nucleic acid base moiety or the 5'- or 3'-terminal hydroxyl groups. As a deprotection reagent, for example, concentrated aqueous ammonia can be used when the protecting group of the phosphate moiety is a cyanoethyl group, or when the protecting group of the nucleic acid base moiety is an acyl protecting group. When the protecting group of the phosphate moiety is an allyl group, a dialkylamide solvent solution containing DABCO and diisopropylamine is exemplified. It is also possible to remove the acyl protecting group from the nucleic acid base moiety using a dialkylamide solvent solution containing DABCO and diisopropylamine. The nitrogen or sulfur nucleophile used for deprotecting the allyl protecting group of the phosphate group is selected from DABCO, trimethylamine, and thiol derivatives; the allyl scavenger is selected from diisopropylamine, diethylamine, imidazole, dodecanethiol, dithiothreitol, and cysteine ​​derivatives; and the reaction solvent is selected from polar solvents such as acetonitrile, DMF, and DBU.

[0047] The purification reagent container 5 contains reagents used to separate and purify the target deprotected product from by-products and excess reagents generated by the two-step deprotection reaction described above.

[0048] The by-products and excess reagents generated by the two-step deprotection reaction described above are separated from the target substance by the separator 8 and collected in the first waste liquid container 7-1. In this embodiment, the separator 8 can be a commonly used one, such as a solid-liquid separator that can separate precipitated oily substances or solids from liquids.

[0049] The target substance container 6 is a container for recovering the oligonucleotide, which is the target substance produced by the purification process described above.

[0050] The second waste liquid container 7-2 is a container for storing waste reagents and impurities separated from the target product by the purification process described above. The third waste liquid container 7-3 is a container for recovering the removed liquid when the solution containing the obtained target product, oligonucleotide, is freeze-dried to form a powder.

[0051] Each of the piping lines from the raw material container 1, the first reagent container 2, the second reagent container 3, the third reagent containers 4-1, 4-2, and the purified reagent container 5 is connected to a liquid delivery device 31 (31-1 to 31-6 in Figure 1) that delivers the solution from each container to the reaction tube connected via the piping. Plunger pumps, diaphragm pumps, syringe pumps, etc., can be used as liquid delivery devices.

[0052] At the first joint 11-1, the raw materials and the first reagent are combined to form a raw material mixture, which is then led to the first reaction tube 21. Within the first reaction tube 21, the raw materials and the activator are mixed and come into contact, causing a reaction for nucleotide chain elongation. The solution containing the chain elongation product is then led to the second reaction tube. Within the second reaction tube, a reaction for oxidation or sulfidation of the chain elongation product takes place.

[0053] The first reaction tube 21, the second reaction tube 22, the third reaction tube 23, and the fourth reaction tube 24 each initiate a reaction by mixing reagents with raw materials or reaction intermediates within their respective reaction tubes. After the reaction at each stage is completed, the completed solution is moved to a fitting or separator located downstream in the flow direction and used for the next reaction. For this reason, the first reaction tube 21, the second reaction tube 22, the third reaction tube 23, and the fourth reaction tube 24 each need to be long enough to complete their respective reactions. In other words, the length of each reaction tube is determined based on the time required to complete each reaction, as well as the flow rate and flow velocity of each reagent solution.

[0054] Furthermore, the amount of reaction that can be completed in each reaction tube also depends on the diameter of the reaction tube. The diameter of the tubes can vary for each reaction tube. For example, tubes with inner diameters of 1 / 8 inch, 1 / 4 inch, and 1 / 2 inch can be used.

[0055] For a 1 / 8 inch tube (2 mm inner diameter), assuming a flow rate of 1 mL / min of solution in the reaction tube, 24 hours of operation would deliver 1.44 L of solution, synthesizing 144 g, resulting in an estimated 1 kg / 7 days. For a 1 / 4 inch tube (4.15 mm inner diameter), assuming a flow rate of 10 mL / min of solution in the reaction tube, 24 hours of operation would deliver 14.4 L of solution, synthesizing 1440 g, resulting in an estimated 1 kg / 16 hours. For a 1 / 2 inch tube (8.5 mm inner diameter), assuming a flow rate of 100 mL / min of solution in the reaction tube, 24 hours would deliver 144 L of solution, synthesizing 14,400 g, resulting in an estimated 1 kg / 1.6 hours.

[0056] Furthermore, to increase the reaction rate, a heater may be provided to warm the reaction tube from all sides.

[0057] The material used for the reaction tube must be one that does not react with the reagents passing through it. Examples include polyfluoroethylene, stainless steel, Teflon, polyethylene, polyvinyl chloride, and PEEK.

[0058] For reactions requiring long reaction times, the reaction tubes become quite long, so they may be coiled up to form a coil.

[0059] The first reagent container 2 contains two or more types of raw materials. One of the raw materials used in this embodiment is a nucleotide building block. The nucleotide building block has, as an example, the structure shown in formula (I) below.

[0060] [ka]

[0061] In formula (I), B is either a natural or non-natural base. 1 , R 2R3 is independently selected from acyl protecting groups such as benzoyl, acetyl, and pivaloyl, as well as acetyl, isobutyryl, and dmf. R3 is independently selected from CH3, CH2CH3, or NR 3 3 forms a ring and is either N(CH2CH2)2 or N(CH2CH2)2. 4 R' is independently one of CH2CH2CN, CH2CH=CH2, CH2(CF2)6CF3, or CH2(CF2)4CF3. X is H, and Y is one of F, OCH3, OMOE, OTBDMS, or forms an XY bond where XY is CH2O, CH2CH2O, CH2NH, CH2NR', or CH2N-N=NH2, where R' is independently one of H, an alkyl group, a carbamate, an amide group, or a substituted silyl group. n is an integer from 0 to 46.

[0062] In a more preferred form, the lower limit of n in formula (I) may be 5, 6, 7, 8, 9, or 10. With such a value, when mixing two types of raw materials, depending on the length and amount of the other raw material used, it is possible to obtain several kilograms to several thousand kilograms of GMP-grade oligonucleotides of 10-mers or more by running the apparatus of this embodiment continuously for one week.

[0063] In a more preferred form, the upper limit of n in formula (I) may be 40, 39, 38, 37, 36, 35, or 34. With such values, when mixing the three raw materials, depending on the length and amount of the other two raw materials used, it is possible to obtain several kilograms to several thousand kilograms of purified oligonucleotides of 100mers or more by running the apparatus of this embodiment continuously for one week.

[0064] As an example of at least one more type of raw material to be contained in the first reagent container 2, another building block can be used in which the hydroxyl group at the 5'-terminus does not have a protecting group, but the hydroxyl group at the 3'-terminus is protected. If this building block has the same type of protecting group as the protecting group used in the nucleotide building block described above on the nucleic acid base portion and the phosphate portion, the deprotection reagent can be made common, which simplifies the process.

[0065] Furthermore, it is possible to contain another type of building block with a different reactivity in the raw material container 1. In this case, the protecting groups of the other building block used are adjusted so that they can react chemoselectively with two other types of building blocks.

[0066] As solvents, acetonitrile, dichloromethane, THF, etc., which are commonly used in the phosphoramidite process, can be used, and a mixture of multiple solvents may also be used. For example, to increase the solubility of the building blocks and products in the solution during the reaction, DMF may be mixed with acetonitrile, dichloromethane, or THF.

[0067] The fitting used in this embodiment may be a valve. To allow the reaction to proceed without stopping the supply of the reagent solution for a predetermined period of time, the valve is always turned ON in the direction of liquid supply. The valve is switched ON / OFF as needed, based on the results of the reaction monitoring described later, or when performing periodic inspections, cleaning, or parts replacement of the equipment.

[0068] To synthesize oligonucleotides that meet the GMP grade requirements for nucleic acid pharmaceuticals, the oligonucleotide continuous synthesis apparatus according to this embodiment is housed in a completely sealed system, such as a cleanroom. Therefore, the startup and monitoring of the apparatus are performed remotely from outside the sealed system. This reduces the number of workers required for synthesis and also shortens the time that workers are exposed to reagents and solvents.

[0069] As described above, in the oligonucleotide continuous synthesis apparatus according to this embodiment, an activator, an oxidizing or sulfiding agent, a deprotection reagent, and a purification reagent are continuously supplied, allowing for continuous chain extension in the first reaction tube, oxidation or sulfidation in the second reaction tube, deprotection reaction in the third reaction tube, and purification in the purification dregs. In other words, the chain extension reaction of the raw materials, through the oxidation or sulfidation reaction and deprotection reaction, and up to the separation and purification process can be continuously executed in a liquid-phase flow manner within a closed system. That is, the oligonucleotide continuous synthesis apparatus according to this embodiment makes it possible to continuously synthesize oligonucleotides from raw materials in a liquid-phase flow manner.

[0070] This allows for the synthesis of GMP-grade oligonucleotides in fewer steps and with less space compared to conventional batch synthesis methods or systems. Furthermore, the reduced number of steps also decreases the number of operations required to obtain the target product, resulting in higher yields and purity compared to commonly used batch synthesis methods.

[0071] Furthermore, shortening the process reduces the amount of waste reagents and waste liquids generated from the solvents and reagents used in the synthesis. This allows for the creation of a synthesis apparatus with a lower environmental impact.

[0072] Figure 1 shows an example of a configuration in which a single device performs freeze-drying of the target oligonucleotide and recovers the oligonucleotide in powder form.

[0073] In the configuration shown in Figure 1, it is also possible to omit the freeze-drying of the target oligonucleotide and instead store the solution of the target product obtained by purification in the purification tank 9 in the product container. In this case, the obtained solution of the target product may be led to the outside of the sealed system of the continuous oligonucleotide synthesis apparatus and freeze-dried in a separate sealed system located outside of that to form a powder. Alternatively, a spray dryer or crystallization may be used instead of freeze-drying for powdering (solidification).

[0074] [Second Embodiment] Figure 2 shows an overview of the configuration of the oligonucleotide continuous synthesis apparatus 200 according to the second embodiment. In Figure 2, components that are denoted by the same reference numerals as those in the first embodiment shown in Figure 1 are the same as those in the first embodiment and their explanation is omitted. The steps taken to obtain the target product from the raw materials are also the same as those in the first embodiment.

[0075] In the configuration of the oligonucleotide continuous synthesis apparatus 200 according to this embodiment, monitoring devices 41, 42, 43, and 44 are arranged between the downstream side of the first reaction tube 21 and the second joint 11-2, between the downstream side of the second reaction tube 22 and the third joint 11-3, between the downstream side of the separator 8 and the purification tank 9, and between the purification tank 9 and the freeze-drying tank 10. It is not necessary for all of these monitoring devices to be present; a configuration with only one, two, or three of them may be provided.

[0076] The monitoring device can be selected from mass spectrometers, NMR, UV detectors, near-infrared spectrometers, Raman spectrometers, conductivity analyzers, etc. For example, in the configuration shown in Figure 2, the monitoring device placed between the downstream side of the first reaction tube 21 and the second joint 11-2 is as follows: 31 P-NMR can be used. Monitoring may be performed by extracting a portion of the reaction solution and using a monitoring device placed outside the apparatus. Alternatively, in-line monitoring may be performed depending on the monitoring device.

[0077] In the configuration shown in Figure 2, for example, the monitoring device 42 placed between the downstream side of the second reaction tube 22 and the third joint 11-3 can be a mass spectrometer. Instead of a mass spectrometer, a UV detector, a near-infrared spectrometer, a Raman spectrometer, or a conductivity analyzer can also be used.

[0078] Similarly, in the configuration shown in Figure 2, a monitoring device 43 is placed between the downstream side of the separator 8 and the purification tank 9, and a monitoring device 44 is placed between the purification tank 9 and the freeze-drying tank 10. 31It may be a P-NMR spectrometer, a UV detector, a near-infrared spectrometer, a Raman spectrometer, or a conductivity measuring device.

[0079] The locations where monitoring devices can be placed are not limited to those shown in Figure 2; additional devices can be placed in different locations as needed. Furthermore, the placement of monitoring devices is not limited to one per location; multiple types of monitoring devices can be placed as required.

[0080] In the oligonucleotide continuous synthesis apparatus according to this embodiment, a monitoring device is placed at least one point immediately after the chain extension step, oxidation or sulfidation step, deprotection step, and purification step. As a result, the target product can be obtained with higher purity compared to a configuration without a monitoring device.

[0081] [Modification 1 of the second embodiment] Figure 3A shows an overview of the configuration of the oligonucleotide continuous synthesis apparatus according to Modification 1 of the second embodiment, in which a monitoring device is installed.

[0082] In the configuration example shown in Figure 3A, the oligonucleotide continuous synthesis apparatus can be separated and reconnected at at least one of the locations indicated by the arrows.

[0083] In the configuration example shown in Figure 3A, a separation tank 61 is positioned between the downstream side of the monitoring device 43, which is located downstream of the separator 8, and the purification tank 9. The separation tank 61 cools the solution containing the target substance obtained from the separator 8, separating the precipitated oily or solid from the supernatant. The supernatant is stored in the purification tank 9, and the precipitated oily or solid is stored in the fourth waste liquid container 7-4.

[0084] Incidentally, in the configuration example shown in Figure 3A, the freeze-drying tank 10 is not located downstream of the monitoring device 44, which is located downstream of the purification tank 9. The solution containing the target substance is stored in the target substance container 6 in its solution state. This target substance solution is then led to another device or facility where freeze-drying is performed on the target substance solution. It is also possible to combine the configuration of the first embodiment with a configuration that stores the target substance solution without passing through the freeze-drying tank 10.

[0085] In the configuration example shown in Figure 3A, the monitoring device 43 is located upstream of the separation tank 61. However, an additional monitoring device may be added downstream of the separation tank 61, or a monitoring device may be placed only downstream of the separation tank 61 without placing one upstream.

[0086] The divided configuration will be explained using specific examples shown in Figures 3B and 3C. The connection relationship between the raw material container 1, the first reagent container 2, and the second reagent container 3, as well as the configuration shown in Figure 3B, in which the first reaction tube 21 for the chain extension process and the second reaction tube 22 downstream for the oxidation or sulfidation process are provided, are common to the first and second embodiments.

[0087] In the configuration shown in Figure 3B, the second reaction tube 22 is connected downstream to a pipe extending from the poor solvent container 71 containing the poor solvent by a third joint 11-7. The downstream side of the third joint 11-7 is connected to the separation tank 61 described above.

[0088] In the configuration shown in Figure 3B, the protected oligonucleotide, which has been elongated in the chain extension step and then had its phosphorous acid portion oxidized or sulfurized in the oxidation or sulfurization step, is led from downstream of the second reaction tube 22 through the third joint 11-3 to the separation tank 61. Therefore, the oxidized or sulfurized protected oligonucleotide led to the separation tank 61 has all the protecting groups and is in the most hydrophobic state in the synthesis process of this embodiment.

[0089] Therefore, by transferring a poor solvent such as alcohol from the poor solvent container 71 to the separation tank 61, the target product, the protected oligonucleotide, precipitates in the separation tank 61 as an oily substance or solid. Since this precipitated oily substance or solid is the target product, it is led to another facility for a deprotection process. In other words, in the configuration example shown in Figure 3A, the oligonucleotide continuous synthesis apparatus is divided at the point indicated by the arrow on the upstream side, and the separation tank 61 is located downstream of that.

[0090] The configuration example shown in Figure 3C is a configuration in which the area between the tip of arrow A and the tip of arrow B is divided from the configuration example shown in Figure 3A, and the separation tank 61 is connected on the downstream side.

[0091] The configuration example shown in Figure 3C involves sequentially deprotecting the protected oligonucleotide (contained in the container 90) obtained in the configuration shown in Figure 3B by reacting it with two types of deprotection reagents, and then separating the resulting deprotected product in the separation tank 61. The target product separated in the separation tank 61 is then led to another facility for a purification process. A commonly used separation tank can be applied, and those using solid-liquid separation, electrodialysis, etc., can also be used.

[0092] Thus, by configuring the oligonucleotide continuous synthesis apparatus according to this embodiment to allow for the separation and reconnection of a portion of it, manufacturing control can be performed for each process. This makes it easier to recover if any part of the process fails.

[0093] [Modification 2 of the second embodiment]

[0094] Figure 4 shows an example of a configuration that includes a deviant container 52 for containing the removed shorter product if monitoring reveals that the product is shorter than the target oligonucleotide.

[0095] In the configuration shown in Figure 4, monitoring is performed by a monitoring device 44 positioned between the purification tank 9 and the freeze-drying tank 10. If the monitoring results show that the product contains offshoots with lengths different from the target oligonucleotide product, a branching connector 51 is installed downstream of the monitoring measures in the synthesis pathway, and the offshoots are collected in an offshoot container 52.

[0096] Monitoring equipment can sometimes reveal the structure of the diverted compound. In such cases, instead of simply discarding the diverted compound as waste, it undergoes necessary purification treatment so that it can be reused as a raw material.

[0097] Thus, by equipping the oligonucleotide continuous synthesis apparatus according to this embodiment with a defect detection device, it becomes possible to separate products that have deviated from the target product from those that are being produced without problems. This allows for maintaining the purity of the crude product of the target product and keeping the load on the purification process constant. Furthermore, by separating and recovering the deviated products, it becomes possible to provide an opportunity to use them again in production.

[0098] Figure 5 shows one specific example of the configuration of the oligonucleotide continuous synthesis apparatus according to the second embodiment.

[0099] The specific examples shown in Figure 5 are mass spectrometers as monitoring devices 41 and 41-3, and monitoring devices 4-2 and 4-4 as 31 It is equipped with a P-NMR spectrum. Furthermore, the first reaction tube 21, the second reaction tube 22, and the third reaction tube 23 are all constructed with the tube wound in a coil shape. However, the configuration is not limited to a coil shape; a flat, bent tube, or other configurations can be selected as appropriate depending on the size of the installation location.

[0100] In this specific example, the outside of the coiled reaction tube is covered with a heating device (for example, an oven). This allows for the application of an appropriate temperature to the reaction in each reaction tube, and the reaction temperature to be maintained at a constant level.

[0101] The mass spectrometers, which are monitoring devices 4-1 and 4-3, extract a portion of the reaction solution from the reaction pathway using a splitter and monitor whether the desired reaction is proceeding and the target product is being obtained.

[0102] In the configuration example shown in Figure 5, the area enclosed by the dotted line is a controlled space configured as a sealed system. Inside this sealed space, the target oligonucleotide, in powder form, is contained in the target container 6. The raw material container, reagent container, etc., are placed outside the sealed system because their size varies depending on the manufacturing scale.

[0103] For example, in the configuration shown in Figure 5, the area enclosed by the dotted line is 1m. 3 It can be installed in a space of approximately 0.6m x 1.2m x 1.8m. The size of the oligonucleotide continuous synthesis apparatus can be changed depending on the purpose, and depending on the size, by operating the apparatus continuously, it is possible to synthesize, for example, 14 kg of oligonucleotides in one day and 100 kg in seven days.

[0104] The diameter of the reaction tube can be determined by the reaction taking place inside it, including its length and diameter. Furthermore, depending on the production scale, it is possible to install a stirrer at the joint where two different solutions are mixed.

[0105] Figure 6 shows one specific example of the configuration of the oligonucleotide continuous synthesis apparatus according to the second embodiment, specifically the configuration related to the supply of liquid from the raw material container 1 and the first reagent container 2.

[0106] Argon gas is blown into the washing solution container 81, the first reagent container 2, the raw material container 1, and the second reagent container 3, and the gas pressure pushes the solutions in each container. As the pumped solutions are used in the reaction and move through the apparatus, the target liquids are sent to the waste liquid and spill recovery containers. Outlets are provided in the waste liquid container 7, spill container 52, and target material container 6, so that the pressure inside the containers remains constant, as does the internal pressure of the reaction tube.

[0107] For example, in an apparatus having the configuration shown in Figure 6, setting the flow rate to 5 mL allows for the synthesis of 6 kg of oligonucleotides per day.

[0108] Figure 7 shows an overview of the connection section in a configuration of a continuous oligonucleotide synthesis apparatus according to the first or second embodiment, in which multiple raw material containers are arranged and pumped. Since the continuous oligonucleotide synthesis apparatus of this embodiment operates continuously, raw materials and reagent solutions are continuously consumed. Therefore, to maintain a continuous supply of raw materials and reagent solutions, multiple raw material containers and reagent containers are arranged and connected to each other. For example, in the case of a raw material container, the remaining amount in the container is monitored during the operation of the apparatus, and if it falls below a predetermined amount, it is necessary to switch to supply from another raw material container. For this reason, the piping that delivers the raw material solution is equipped with a switch from each raw material container. In the configuration example shown in Figure 7, the liquid is delivered by a pump. Pump delivery is preferred in large-scale synthesis.

[0109] Figure 8 shows an overview of the connection section in a configuration of a continuous oligonucleotide synthesis apparatus according to the first or second embodiment, in which multiple raw material containers are arranged and supplied by gas. Similar to the configuration in Figure 7, multiple containers are arranged and connected. In the configuration in Figure 8, the liquid is supplied by gas pressure, and gas is supplied to each container via a manipulator. During operation of the apparatus, the remaining amount in the containers is monitored, and if it falls below a predetermined amount, a switch switches to supply from another raw material container. The gas pressure supply method is preferred for small to medium-scale synthesis. [Examples]

[0110] [Example 1] A tube was used as the reactor, and the chain extension process was tested. 2.6 mL of a 20 mM dichloromethane-DMF (9:1) solution of DMTr-dTCGTCGTTTTGT 3'-phosphoramidite (12-mer A, 279 mg, 5 μmol, G is an amino group protected with isobutyryl groups, all phosphate groups protected with allyl groups, sulfurized) and HO-dCGTTTTGTCGTT-dca (12-mer B, 233 mg, 49 μmol, G is an amino group protected with isobutyryl groups, all phosphate groups protected with allyl groups, sulfurized) and a 55 mM dichloromethane solution (0.4 mL) of 5-ethylthio-1H-tetrazole (28 mg, 216 μmol) were delivered into tetrafluoroethylene tubes with an inner diameter of 1 mm at flow rates of 5 mL / min and 0.8 mL / min, respectively, and allowed to remain for 30 minutes. The reaction solution was extracted and subjected to HPLC analysis. The absorption intensity at 260 nm was used as an indicator, and the consumption rate of the two raw materials and the yield of the 24-mer product were confirmed from the area ratio. In four experiments, the consumption rate of dodecamer A was over 99%, and the yield of the resulting 24-mer was 83-88% in all cases.

[0111] [Example 2] Using a tube as the reactor, the sulfidation process of the 24-mer (one phosphate group being phosphorous acid) obtained in Example 1 was tested. A 20 mM dichloromethane-DMF (9:1) solution (2.6 mL) of the 24-mer DMTr-dTCGTCGTTTTGTpCGTTTTGTCGTT-dca (233 mg, 49 μmol) containing the phosphorous acid ester, and a 100 mM acetonitrile solution (0.6 mL) of the sulfidating agent 5-phenyl-1,2-dithiazol-3-one (12 mg, 60 μmol), were delivered into a tetrafluoroethylene tube with an inner diameter of 1 mm at flow rates of 5 mL / min and 1 mL / min, respectively, and allowed to stand for 30 minutes. The reaction solution was removed and subjected to HPLC analysis, and the absorption intensity at 260 nm was used as an indicator. The consumption rate of the phosphorous acid raw material and the yield of the sulfidized phosphoric acid product DMTr-dTCGTCGTTTTGTpsCGTTTTGTCGTT-dca were confirmed from the area ratio. In four experiments, the results showed that the phosphite consumption rate was 99% or higher and the sulfide yield was 99% or higher in all cases.

[0112] [Example 3] Using a tube as a reactor, a precipitation test of the 24 sulfide obtained in Example 2 was conducted. Ethyl acetate (3.6 mL) was added to the sulfurization reaction solution (3.6 mL) from Example 2, and the resulting precipitate was dried to obtain the target 24 sulfide. In three trials, the recovery rate was slightly over 80%, and the purity averaged 73%, indicating that some impurities from the reagent and the dodecamer raw material were removed. This confirmed that the process can be used for semi-continuous production.

[0113] By performing the above precipitation process three times, an improvement in purity was confirmed.

[0114] [Table 1]

[0115] [Example 4] Deprotection tests were conducted on the 24-sulfide obtained in Example 3. The optimization of the allyl group deprotection reaction conditions was investigated using the 24-sulfide. As a result, it was found that the system using DABCO as the deallylizing agent, diisopropylamine as the allyl scavenger, and a dialkylamide solvent was the best.

[0116] The entire amount of the sulfide 24-mer obtained in the previous step was dissolved in a 2% dichloroacetic acid dichloromethane solution (4 mL) and treated at room temperature for 30 minutes to remove the DMTr group. The reaction mixture was added dropwise to saturated sodium bicarbonate aqueous solution (20 mL), and the organic layer obtained by liquid-liquid extraction was added dropwise to ethyl acetate (20 mL) to obtain the DMTr-free 24-mer as a colorless powder. This was treated under the conditions of entry 4, in a 2% DABCO DMF solution (w / v, 17.9 mL) at 50 °C for 10 hours, and the treated solution was added dropwise to 170 mL of n-hexane-ethyl acetate (3:7) solution to obtain the allyl-free 24-mer as a colorless powder. The acyl group was removed by further treatment with 28% ammonia aqueous solution (v / v, 17.9 mL) at 50 °C for 10 hours. Ammonia was removed under reduced pressure, and the crude product purity of the target 24-mer HO-dTCGTCGTTTTGTCGTTTTGTCGTT-OH was confirmed by HPLC. A series of deprotection operations were performed on three samples, and it was confirmed that the yield and purity were consistent (Table 2). The HPLC chart of the crude 24-mer product obtained in Entry 2 is shown in Figure 9. The peak at a retention time of 13.58 minutes represents the crude 24-mer product, the peaks at retention times of 10.13 minutes and 12 minutes represent impurities derived from the dodecamer starting material, and the peak at a retention time of 4.65 minutes represents impurities derived from the freed protecting group. [Table 2]

[0117] In this deprotection process, in a continuous production system, the DMTr group is replaced with an acyl protecting group such as a benzoyl group, canceling the dichloroacetic acid treatment for DMTr group removal, and the deallylization and deacylization processes are executed in a single process under the conditions of entry 6.

[0118] [Example 5] The purification process for the 24-mer obtained in Example 4 was tested. The crude product solution could be purified to obtain a high-purity 24-mer product. It was confirmed that a continuous process is possible in a flow synthesis apparatus by employing membrane separation purification.

[0119] Ultrafiltration membrane separation: Purification was performed using AmiconUltra10k (Merck) under the following conditions.

[0120] Ultrafiltration membrane separation (TFF method): Separation membrane: Amicon Ultra10k (Merck) Column volume: 1 mL Sample: 0.5 mg / mL, 500 μL Diluent: 25 mM NaOH aqueous solution Centrifugation conditions: 14000g, 15 minutes x 3 times Recovery rate: 75% Purity (HPLC A260 integrated value): 95.75% The HPLC chart of the purified 24-mer product is shown in Figure 10.

[0121] (Gel filtration purification) Purification was performed under the following conditions. Gel: P-60 (45-90μm) / BioRad Column size: 1.3cm ID x 11cm Column volume: Approximately 10 mL Sample: 1 mg / mL, 300 μL, 3% of column volume Equilibration eluate: 0.1M TEAA buffer (pH7.0) Amount: 2mL (Fr.1-2, 10), 1mL (Fr.3-9) Flow rate: Natural fall (approx. 0.166ml / min) Recovery rate: 73% Purity (HPLC A260 integrated value): 95.41% The HPLC chart after purification is shown in Figure 11.

[0122] (ODS filtration) Purification was carried out under the following conditions. The fraction containing impurities was re-filtered three times to obtain the target product with a 70% recovery rate and an HPLC purity (A260) of 97.4%. Column: YAMAZEN ODS-SM 50μm 120Å L (1CV=72mL) Eluent A: Elix Water B: Acetonitrile Gradient: 13%B (0-6.20 min), 100%B (6.21-15 min) Flow rate: 20mL / min Temperature: ambient Detection: UV (254nm) Sample volume: Approximately 496 mg, total volume of 20 mL (1 / 4 CV volume) The HPLC chart after purification is shown in Figure 12. The peak around the 15-minute retention time represents the product conformer, which is included in the purity.

[0123] (Precipitation) The product was precipitated by adding alcohol to a deprotection solution containing ammonia under the following conditions. Sample: Ammonia-treated solution containing 16 mg / mL of ammonia. The precipitation conditions are as follows: [Ethanol precipitation] 24-mer solution: Ethanol: 3M sodium acetate = 1:2.5:0.1 100 μL: 250 μL: 10 μL Temperature: -20℃ Product recovery rate: 93% [IPA precipitation] 24-mer solution: IPA:3M sodium acetate = 1:1:0.1 100 μL: 100 μL: 10 μL Temperature: -20℃ Product return rate: 84% The HPLC chart of the purified 24-mer product is shown in Figure 13. [Explanation of Symbols]

[0124] 1. Raw material container 2. First reagent container 3. Second reagent container 4(4-1,4-2) Third reagent container 5. Container for purified reagents 6. Container for the object 7 (7-1, 7-2, 7-3, 7-4) Waste liquid container 8 Separators 9 Purification tank 10 Freeze drying tank 11(11-1,11-2,11-3,11-4…,11-7) Fittings 21 First reaction tube 22 Second reaction tube 23 Third reaction tube 24 Fourth reaction tube 31 (31-1, 31-2, ..., 31-9) Liquid delivery device 41, 42, 43, 44 Monitoring devices 51 Deviation device 52. Container for contained deviant material 61 Separation tank 71 Poor Solvent Container 81. Washing solution container 100, 200, 300, 310, 320, 400, 500 Oligonucleotide Continuous Synthesis System

Claims

1. A raw material container containing a mixture of at least two types of protective nucleoside block bodies, which are the raw materials, A first reagent container containing a first reagent for reacting at least two of the aforementioned protected nucleoside block bodies with each other to extend the nucleotide chain, A second reagent container for a reaction to oxidize or sulfurize the phosphate bond of the nucleotide elongated product obtained by the chain elongation, A third reagent container for a reaction to deprotect the nucleotide elongated product that has been oxidized or sulfurized, A first joint connecting the piping extending from the raw material container and the piping extending from the first reagent container, A first reaction tube connecting the first fitting and a second fitting connected to piping extending from the second reagent container, A second reaction tube connecting the second fitting and a third fitting connected to piping extending from the third reagent container, Equipped with, The raw materials, the first reagent, the second reagent, and the third reagent are each in solution. A liquid delivery device is connected to each of the piping from the raw material container, the first reagent container, the second reagent container, and the third reagent container, which delivers the solution from each container to the reaction tube connected through the piping. At the first joint, the raw material solution and the first reagent solution are combined to form a raw material mixture, which is then led to the first reaction tube. In the first reaction tube, the reaction for chain elongation of the nucleotide is carried out using the raw material mixture, and the solution containing the chain elongation product is introduced into the second reaction tube. In the second reaction tube, the oxidation or sulfidation reaction of the chain extension product is carried out. A continuous oligonucleotide synthesis apparatus that continuously synthesizes oligonucleotides by continuously supplying the raw materials, the first reagent, and the second reagent.

2. The system further comprises a first waste liquid container for containing waste liquid containing waste reagents generated by the chain extension reaction, the oxidation or sulfidation reaction, and the deprotection reaction, The third joint and the separator connected to the piping extending from the first waste liquid container are connected to the third reaction tube. The oligonucleotide continuous synthesis apparatus according to claim 1, wherein the deprotection reaction is carried out in the third reaction tube and led to the separator, the separator separates the crude oligonucleotide product obtained by the chain extension reaction and the oxidation or sulfidation reaction from the waste reagents generated in each reaction, and the waste liquid containing the waste reagents is contained in the first waste liquid container.

3. A purification reagent container for purifying the crude oligonucleotide product, A container for the purified oligonucleotide, which is the target product, A second waste liquid container for containing waste liquid containing waste reagents generated in the aforementioned purification process, Furthermore, A liquid delivery device is connected to the piping from the aforementioned purified reagent container, which delivers the solution from the purified reagent container through the piping. The oligonucleotide continuous synthesis apparatus according to claim 2, wherein the separator, the piping extending from the purification reagent container, and the piping extending from the second waste liquid container are connected to a purification tank, the crude oligonucleotide product is purified in the purification tank, and the final oligonucleotide product obtained from the downstream side of the purification tank is contained in the target product container, thereby carrying out the reaction for chain extension and the purification of the crude oligonucleotide product continuously in the liquid phase, and synthesizing the final oligonucleotide product continuously from the raw materials.

4. The oligonucleotide continuous synthesis apparatus according to claim 3, wherein the purification tank and piping extending from the target material container are connected to a freeze-drying tank, in which the purified oligonucleotide is concentrated and dried to form a powder, and the powdered oligonucleotide is contained in the target material container.

5. The oligonucleotide continuous synthesis apparatus according to claim 1, wherein a monitoring device is positioned at least one of the following locations: between the downstream side of the first reaction tube and the second joint, or between the downstream side of the second reaction tube and the third joint.

6. The oligonucleotide continuous synthesis apparatus according to claim 3, wherein a monitoring device is arranged between the downstream side of the separator and the purification tank.

7. The oligonucleotide continuous synthesis apparatus according to claim 4, wherein a monitoring device is arranged between the purification tank and the freeze-drying tank, and a fitting is arranged between the monitoring device and the freeze-drying tank to connect to a defect recovery device for recovering defect products of a different length from the target oligonucleotide product.

8. The oligonucleotide continuous synthesis apparatus according to any one of claims 5 to 7, wherein the monitoring device is selected from a mass spectrometer, NMR, UV-visible light spectrometer, near-infrared spectrometer, Raman spectrometer, conductivity measuring device, and pH measuring device.

9. The oligonucleotide continuous synthesis apparatus according to any one of claims 5 to 7, wherein the monitoring device is divisibly connected downstream.

10. The oligonucleotide continuous synthesis apparatus according to claim 1, wherein two types of the protective nucleoside block bodies are mixed and contained in the raw material container.

11. The oligonucleotide continuous synthesis apparatus according to claim 1, wherein three types of the protective nucleoside block bodies are mixed and contained in the raw material container.

12. The oligonucleotide continuous synthesis apparatus according to claim 3, wherein the reaction for chain extension, the reaction for oxidation or sulfidation, the reaction for deprotection, and the purification are carried out continuously in the liquid phase.

13. The oligonucleotide continuous synthesis apparatus according to claim 2, wherein at least one of the first reaction tube and the third reaction tube is a tube in which a catalyst used for the reaction is fixed inside.

14. A method for synthesizing oligonucleotides using the oligonucleotide continuous synthesis apparatus described in claim 3, A chain elongation step in which nucleotide chain elongation is performed by reacting at least two types of protected nucleoside block bodies with each other, An oxidation or sulfurization step is performed to oxidize or sulfurize the phosphate bond portion of the nucleotide elongation obtained in the chain elongation step, A deprotection step is performed on the nucleotide elongated product obtained in the oxidation or sulfidation step, A purification step for purifying the crude oligonucleotide product obtained in the deprotection step, Equipped with, A method for synthesizing oligonucleotides, wherein the chain extension step, the oxidation or sulfurization step, the deprotection step, and the purification step are carried out sequentially in a liquid phase.

15. The method for synthesizing oligonucleotides according to claim 14, wherein the number of nucleosides contained in one of the protective nucleoside block bodies used in the chain extension step is in the range of 1 to 48.

16. A method for synthesizing an oligonucleotide of a desired chain length by condensing two types of protected nucleoside block bodies, as described in claim 14.

17. A method for synthesizing oligonucleotides according to claim 14, wherein the target oligonucleotide is purified by precipitation or by oil-out.

18. A method for synthesizing oligonucleotides according to claim 14, wherein the allyl protecting group of the phosphate portion is deprotected by removing it with nitrogen or a sulfur nucleophile, the nitrogen or sulfur nucleophile is selected from DABCO, trimethylamine, and thiol derivatives, the allyl scavenger is selected from diisopropylamine, diethylamine, imidazole, dodecanethiol, dithiothreitol, and cysteine ​​derivatives, and the reaction solvent is selected from acetonitrile, DMF, and DBU.

19. A method for synthesizing oligonucleotides according to claim 14, wherein the final oligonucleotide product is obtained by precipitation from a deprotection solution.

20. A method for synthesizing oligonucleotides according to claim 14, comprising obtaining the final oligonucleotide product by precipitation from a deprotection solution, followed by filtration or membrane separation.

Images